First report of leaf spot on Rhododendron delavayi caused by Pestalotiopsis scoparia in China

Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China (Cai et al. 2015). From May to July in 2022, symptoms of leaf spot were observed on R. delavayi over a wide portion of the Baili Azalea Forest Area (N 27°10'-27°20', E 105°04'-106°04'), Guizhou Province, China. About 500 plants were surveyed and the incidence of leaf spot on R. delavayi leaves was 20 to 30%, significantly reducing their ornamental and economic value. The affected leaves had irregular, dark brown lesions with a clear blackish brown boundary and black conidiomata in a grayish center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GUDJ 61, GUDJ 62, GUDJ 63) that produced identical in morphology were obtained. The isolate GUDJ 61 was used for further study. Colonies on PDA grew velvety white on the upper surface and light yellow on the lower surface. Conidia were 5-celled, spindle- to ellipsoid-shaped, straight or slightly curved, 4-septate, and measured 39.0 ± 3.7 × 10.4 ± 0.79 µm (n=50). The morphological features were consistent with the description of Pestalotiopsis scoparia Maharachch., K.D. Hyde & Crous, (Maharachchikumbura et al. 2014). The pathogen was confirmed to be P. scoparia by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), and the partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b, and EF1-728F/EF-2, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers OP048045 (ITS), OP058111 (TUB) and OP058114 (TEF), respectively. BLAST searches of the sequences revealed 99% (549/552 nt), 99% (711/714 nt), and 82% (130/158 nt) homology with those of P. scoparia CBS 176.25T form GenBank (KM199330, KM199393, and KM199478), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUDJ 61 in a well-supported cluster with P. scoparia. The pathogen was thus identified as P. scoparia based on the morphological characterization and molecular analyses. The pathogenicity of GUDJ 61was tested through a pot assay. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GUDJ 61 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times (Fang 2007). After 12 days, the treated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. delavayi caused by P. scoparia in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. delavayi.